Glycoproteins, covalent conjugates of carbohydrates and proteins, are essential in maintaining and regulating intracellular and extracellular biological activities in a living organism. The external surface of the cellular plasma membrane is enriched with glycoproteins which function as cell surface receptors for growth factors, hormones and toxins. In addition, glycoproteins secreted into extracellular fluids exist as growth factors, hormones, enzymes and antibodies. The effect of the carbohydrate moieties on the biological activity of glycoproteins is a subject of continuing studies. It has been shown that carbohydrate moieties play a vital role in regulating the structure and function of a glycoprotein. The carbohydrate moieties may mediate the cellular uptake of the protein, protect the protein from denaturation and proteolytic degradation, or modulate physical properties of the protein such as viscosity, stability, circulatory lifetime and binding capacity (Experientia (1982) 38, pp. 1129-1162; and Ann. Rev. Biochem. (1988) 57, pp. 785-838). Recent studies also demonstrated the ability of the oligosaccharide moieties of glycoproteins to reduce self-association of the proteins (Biochem. (1990) 29, pp. 2482-2487).
Glycosylation is a biologically important modification of the covalent structure of a protein. There are two categories of glycosylation recognized in most eukaryotic systems: O-linked glycans are attached to proteins via an .alpha.-glycosidic linkage to either serine or threonine, while the more frequent N-linked glycans are covalently attached via .beta.-glycosidic bond to an asparagine moiety. Protein N-glycosylation occurs co-translationally in the endoplasmic reticulum with the oligosaccharide Glc.sub.3 Man.sub.9 GlcNAc.sub.2 being transferred en bloc from the lipid carrier dolichol diphosphate to an asparagine residue contained in the peptide sequence of Asn-X-Ser(Thr). Subsequent processing of the nascent glycoprotein in the Golgi by a battery of glycosidases and glycosyltransferases gives rise to a myriad of possible structures for the mature N-linked glycan. Many literature reviews of the structure, function and biosynthesis of N-linked glycans have been recently published (Baenzinger in The Plasma Proteins (1984) IV pp. 271-315; Snider in Biology of Carbohydrates (1984) 2, pp. 164-198; and Kornfeld and Kornfeld in Ann. Rev. Biochem. (1985) 54 pp. 631-664).
A necessary step in obtaining information concerning N-glycan structure, function and biosynthesis is the development of synthetic methodology for the preparation of specific asparagine N-linked glycans, as well as the oligosaccharide dolichol diphosphate lipid intermediate and other carbohydrate derivatives.
Syntheses of all these glycosides is dependent on the chemical "activation" of the terminal GlcNAc moiety of the glycan. One successful glycosyl activation strategy involves the acid-catalyzed formation of an oxazoline intermediate from peracetylated oligosaccharides. For instance, Warren et al. in International Patent Application PCT/U587/01832 describe the synthesis of a high-mannose oligosaccharide asparagine derivative, from an oxazoline derivative and Warren et al. (Carbohydr. Res. (1984) 126 pp. 61-80) also describe the use of an oxazoline to prepare the dolichol diphosphate lipid intermediate.
The synthetic methodology utilized by Warren et al. in the preparation of asparagine-linked high-mannose oligosaccharide derivatives was based on a three-step protocol: (1) addition of azide to the oxazoline intermediate so as to obtain the .beta.-linked azido derivative, (2) reduction of the .beta.-azide to a .beta.-1-amino glycoside and (3) coupling of the glycosylamine to the appropriately protected aspartate moiety. The general synthesis of Asn-linked oligosaccharides is thus ultimately dependent on a mild and efficient method for oxazoline generation. The Asn-linked oligosaccharides are of importance because they may be incorporated into N-linked glycopeptides by condensation of various peptides to the glycosylated asparagine amino-acid moiety. These N-linked glycopeptides have potential use, not only as glycosidase substrate analogues, but also as model systems for studying the structure and dynamics of N-linked glycoproteins.
Peracetyl oxazolines of mono- or disaccharides are traditionally prepared via chloroacetolysis (acetyl chloride in concentrated HCl) of the corresponding peracetyl saccharide. Warren et al. (Carbohydr. Res. (1977) 53 pp. 67-84, (1980) 92 pp. 85-101, and (1984) 126 pp. 61-80) describe the preparation of the peracetylated oxazolines of various saccharides. A severe problem associated with the chloroacetolysis procedure is the susceptibility for cleavage of any acid-labile glycosidic bonds. For instance any .alpha.-D (1.fwdarw.6) glycosidic linkages in the particular oligosaccharide and the .beta.D(1.fwdarw.4) linkages between the two N-acetylglucosamines (GlcNAc) of the oligosaccharide's chitobiose core are particularly prone to acid-catalyzed glycosidic cleavage. The relatively harsh conditions of the chloroacetolysis reaction are therefore not amenable to many structurally complex oligosaccharides.
The direct formation of a peracetyl oxazoline from the peracetate using a Lewis acid as catalyst is an alternative approach to the chloroacetolysis method. Matta et al. in Carbohydr. Res. 1973) 26 pp. 215-218 describe the formation of an oxazoline from the .beta.-D-anomer of peracetyl saccharides using anhydrous ferric chloride. Ferric chloride does not, however, catalyze formation of the oxazoline from the predominant .alpha.-D-anomer of the peracetyl saccharide. Because of its inability to promote oxazoline formation from the major anomer the ferric chloride method is of limited applicability.
Scrivastava describes a method, using ferric chloride, for oxazoline generation from the .alpha.-D-anomer of peracetyl glucosamine (Carbohydr. Res. (1982) 103 pp. 286-292). When this method was applied to peracetyl oligosaccharides the predominance of side reactions led to low yields of desired product. Nakabayashi et al., in Carbohydr. Res., (1986) 150 C7-C10 and Warren et al., in International Patent Application PCT/U587/01832 describe an alternative Lewis-Acid catalyzed procedure for oxazoline generation. This method is designed to circumvent the two aforementioned problems of poor anomeric selectivity and low overall yields of oxazoline. Using one equivalent of the Lewis acid trimethylsilyl trifluoromethanesulfonate these authors formed a peracetyl oxazoline from a heptasaccharide peracetate isomer (Man.sub.5 GlcNAc.sub.2). Nakabayashi et al. claimed that the heptasaccharide oxazoline was accessible from either the .alpha. or .beta. anomer, and they also reported that no glycosidic bond cleavage was detected.
Activated oligosaccharide derivatives may be reacted with proteins to form oligosaccharide-protein conjugates (neoglycoproteins). Because oligosaccharides have a wide spectrum of biological activity, the formation of neoglycoproteins is valuable for improving the biological and physiochemical activity of proteins. Marburg et al. reported a method of preparing oligosaccharide conjugates wherein oligosaccharides are activated by the addition of carbonyldiimidazole or nitrophenyl chloroformate and then reacted with a spacer group with a pendant electrophilic group followed by reaction with a nucleophilic group on a protein (U.S. Pat. No. 4,830,852 (1989)). Some neoglycoproteins have improved thermal stability and increased stability towards proteases and denaturants. For example, after incubation at 60.degree. C. for 10 min, lactosylated E. coli L-asparaginase retains 63% of its activity, whereas the native enzyme retains only 19%. The same modification is also reported to stabilize the asparaginase towards proteolytic digestion. Lactosylated asparaginase retains 50% of its activity after a 60 min incubation with subtilisin, whereas the native asparaginase is completely deactivated after only 20 min incubation (J. Biol. Chem. (1977) 252 pp. 7678.) In addition, neoglycoproteins often have longer circulatory lifetimes. For example, dextran-amylase conjugates have a circulatory lifetime which is 4.5 times longer than the unmodified enzyme (Trends Biochem. Sci. (1978) 3 pp. 79.)
Another interesting aspect of neoglycoproteins is their potential in efficient drug delivery, especially in targeting therapeutic proteins to particular cells or organelles. Endocytosis of glycoproteins is often mediated by the interaction of cell-surface receptors and the glycoprotein carbohydrate components. The attachment of the appropriate carbohydrate components could result in the delivery of neoglycoproteins to specific tissues or organs of the body. For example glycoproteins with terminal galactose are selectively removed from circulation by hepatocytes (Adv. Enzymol. Relat. Areas Mol. Biol. (1978) 41 pp. 99-128). In another example, by attaching lactose to the bovine pancreatic RNAse A dimer, the uptake of the protein was shifted from the kidney to the liver, where galactosyl receptors are located (J. Biol. Chem. (1978) 253, pp. 2070-2072). It has also been shown that the binding affinity of mannosylated bovine serum albumin to the mannose-receptors on rabbit alveolar macrophages increases dramatically, even with a small increase in the number of mannose attached (Biochemistry, (1988) 15 pp. 3956.) Thus, neoglycoproteins hold great therapeutic potential, especially in the treatment of diseases caused by enzyme deficiencies.
There have been numerous methods reported in the literature for the covalent coupling of carbohydrates to proteins (Advances in Carbohydrate Chemistry and Biochemistry (1980) 37 pp. 225-281; CRC Critical Reviews in Biochemistry (1981) pp. 259-306; Glycoconjugates (1982) IV pp. 57-83). For example, p-aminophenyl glycosides of the oligosaccharide may be diazotized and allowed to react with lysyl, tyrosyl and histidyl residues of the protein. Alternatively, the p-aminophenyl glycoside may be converted to an isothiocyanate and attached to amino functional groups on the protein. Another method of neoglycoprotein production involves amidation between a carboxy group and an amine facilitated by dicyclohexylcarbodiimide (DCC), 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide (DAEC) or mixed anhydrides. The carboxy component may be the protein's side-chain carboxy groups or aldonic acids generated by oxidation of the terminal oligosaccharide residue. The amino component may be aminoglycosides or lysines of the protein. Hydrazido-oligosaccharides derivatives may be converted by treatment with nitrous acid into acyl azides, which are highly reactive towards lysine residues of proteins.
Direct coupling of carbohydrates to proteins may also be achieved by reductive amination. In this procedure, the reducing terminus of the oligosaccharide is allowed to react with the amino groups of the protein to form a Schiff base; the Schiff base is subsequently reduced with sodium cyanoborohydride, providing a hydrolytically stable amine linkage between carbohydrate and protein. In general, there is a current need for efficient and specific methods which allow coupling of proteins and oligosaccharides.